Above and belowground phenotypic response to exogenous auxin across Arabidopsis thaliana mutants and natural accessions varies from seedling to reproductive maturity

Background Plant hormones influence phenology, development, and function of above and belowground plant structures. In seedlings, auxin influences the initiation and development of lateral roots and root systems. How auxin-related genes influence root initiation at early life stages has been investigated from numerous perspectives. There is a gap in our understanding of how these genes influence root size through the life cycle and in mature plants. Across development, the influence of a particular gene on plant phenotypes is partly regulated by the addition of a poly-A tail to mRNA transcripts via alternative polyadenylation (APA). Auxin related genes have documented variation in APA, with auxin itself contributing to APA site switches. Studies of the influence of exogenous auxin on natural plant accessions and mutants of auxin pathway gene families exhibiting variation in APA are required for a more complete understanding of genotype by development by hormone interactions in whole plant and fitness traits. Methods We studied Arabidopsis thaliana homozygous mutant lines with inserts in auxin-related genes previously identified to exhibit variation in number of APA sites. Our growth chamber experiment included wildtype Col-0 controls, mutant lines, and natural accession phytometers. We applied exogenous auxin through the life cycle. We quantified belowground and aboveground phenotypes in 14 day old, 21 day old seedlings and plants at reproductive maturity. We contrasted root, rosette and flowering phenotypes across wildtype, auxin mutant, and natural accession lines, APA groups, hormone treatments, and life stages using general linear models. Results The root systems and rosettes of mutant lines in auxin related genes varied in response to auxin applications across life stages and varied between genotypes within life stages. In seedlings, exposure to auxin decreased size, but increased lateral root density, whereas at reproductive maturity, plants displayed greater aboveground mass and total root length. These differences may in part be due to a shift which delayed the reproductive stage when plants were treated with auxin. Root traits of auxin related mutants depended on the number of APA sites of mutant genes and the plant’s developmental stage. Mutants with inserts in genes with many APA sites exhibited lower early seedling belowground biomass than those with few APA sites but only when exposed to exogenous auxin. As we observed different responses to exogenous auxin across the life cycle, we advocate for further studies of belowground traits and hormones at reproductive maturity. Studying phenotypic variation of genotypes across life stages and hormone environments will uncover additional shared patterns across traits, assisting efforts to potentially reach breeding targets and enhance our understanding of variation of genotypes in natural systems.


Supplemental Materials
Supplement S1: Additional data from publicly available databases on variation in APA of genes selected for study.We gathered genome annotation data from Araport11 (Cheng et al. 2017)  Multiple inserts within one annotation represent multiple inserts detected at the SALK institute.
The lines used for this investigation, however, have been confirmed by unPAK (See Rutter et al. 2019) to be homozygous single inserts.

Supplement S2: Synthesis of Existing Auxin Root Assay Literature
We searched the Web of Science database in September 2020 using the search term "lateral root development gene auxin" to obtain articles studying the role of auxin in lateral root development in the window from 1980 -Sept 2020.We then completed a set of filters (Figure S1) based on a series of criteria.Papers were retained if they mentioned lateral roots within the abstract and if they noted the specific loci of genes studied.Our goal for quantitative synthesis of publications was to determine the developmental stage at which the genes frequently studied in the literature, the substrate in which plants were grown, the age at which roots were phenotype, and the method and traits used for root phenotyping.
The results of this synthesis informed our methods by identifying gaps in the literature in the developmental stage of root assays.Lateral root traits of auxin-related mutants were measured after a mean of 11 days (SD=5.0;Fig. S3), with 94% of studies growing plants on an agar substrate (Fig. S4).These findings guided our choices in determining when plants were examined (14d, 21d, and maturity) and the substrates on which they were grown (sand and potting mix).S5 -Analysis of variance examining the fixed effects of hormone treatment, genotype, and their interaction in mature SALK insert mutants.Values are F-statistic results of linear models, and significance levels are indicated by p<0.001=***, p<0.01=**, p<0.05=*, p>0.05=ns with exact p-values noted in parentheses.Inflorescence height has been logtransformed and fruit number, days to bolt, and days to flower square root transformed to meet assumptions.The tray random effect term refers to plant plug trays and was included to account for random differences across our block design.Likelihood ratio test statistics are reported for random effects with exact p-values in parentheses.S8 -Analysis of Variance examining the fixed effects of hormone treatment, genotype, and their interaction in mature plants.All traits have been scaled according to their developmental stage and treatment such that developmental stage*genotype effects represent changes in rank order of genotypes but not different magnitudes or direction of difference.
Values are F-statistic results of linear models, and significance levels are indicated by p<0.001=***, p<0.01=**, p<0.05=*, p>0.05=ns with exact p-values noted in parentheses.The tray random effect term refers to plant plug trays and was included to account for random differences across our block design.Likelihood ratio test statistics are reported with exact pvalues in parentheses.
, polyadenylation cluster location data from the PlantAPAdb (Zhu et al. 2020), and T-DNA insert location data from T-DNA Express (signal.salk.edu) to visualize the variation across loci of interest.

Figure S1 -
Figure S1 -Annotated transcripts of focal genes in the study.Titles of plots include the gene name, its locus, and corresponding SALK insert mutant line available in TAIR11.Different plots underneath the same gene, locus, and SALK line represent different transcripts described in the Araport11 genome annotation.Pink circles represent polyadenylation clusters recognized by studies within the PlantAPAdb.Pink lines represent the location of SALK T-DNA inserts.

Figure S2 -
Figure S2 -Flow chart diagram of literature identification, screening and selection adapted from (Duenas et al. 2021) based on PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines (Moher et al. 2009).

Figure S3 -
Figure S3 -Time of lateral root measurements noted in the publications from the quantitative synthesis of the literature (n=166, mean days old plant = 10.8, +/-1 SD=5.03).

Figure S5 -
Figure S5 -Distribution of major growth traits in both treatments (control in A and auxin in B) across all life stages (14d, 21d, and mature adult left to right) for insert mutants (pink) and natural accessions (blue).Significant difference in variation between natural accessions and insert mutants determined via Levene's tests are noted by an * (df = 1, F value = 3.98, p=0.0481).

Table S2 -Analysis of variance examining the fixed effects of hormone treatment, genotype, and their interaction in 14d SALK insert mutant seedlings.
Values are F-statistic results of linear models, and significance levels are indicated by p<0.001=***, p<0.01=**, p<0.05=*, p>0.05=ns with exact p-values noted in parentheses.Belowground mass and LR density have been square root transformed and aboveground mass log-transformed to meet ANOVA assumptions.

Table S4 -Analysis of variance examining the fixed effects of hormone treatment, genotype, and their interaction in 21d SALK insert mutant seedlings.
Values are F-statistic results of linear models, and significance levels are indicated by p<0.001=***, p<0.01=**, p<0.05=*, p>0.05=ns with exact p-values noted in parentheses.Belowground mass, aboveground mass, and LR density have been square root transformed and rosette diameter log-transformed to meet assumptions.

Table S6 -Mean and one standard error of traits measured in mature adult plants in control and auxin hormone treatments.
Bold mean values indicate a significant difference between treatments.

Table S7 -Analysis of variance examining the fixed effects of genotype, and developmental stage by genotype interaction for 14d and 21d seedlings.
Values are F-statistic results of linear models, and significance levels are indicated by p<0.001=***, p<0.01=**, p<0.05=*, p>0.05=ns with exact p-values noted in parentheses.Belowground mass, aboveground mass, and LR density have been square root transformed and rosette diameter as well as root length have been logtransformed to meet assumptions.Likelihood ratio test statistics are reported for random effects with exact p-values in parentheses.

Table S9 -Analysis of Variance examining the fixed effects of developmental stage, APA group (0, 1+2, or >2) and their interaction in mature SALK insert mutants.
All traits have been scaled according to their developmental stage and treatment such that developmental stage*APA group effects represent changes in rank order of APA groups but not different magnitudes or direction of difference.Values are F-statistic results of linear models, and significance levels are indicated by p<0.001=***, p<0.01=**, p<0.05=*, p>0.05=ns with exact p-values note in parentheses.The tray random effect term refers to plant plug trays and was included to account for the block design.Likelihood ratio test statistics are reported with exact pvalues in parentheses.